1. Crystal Structure of Human Profilaggrin S100 Domain and Identification of Target Proteins Annexin II, Stratifin, and HSP27 
Christopher G. Bunick, Richard B. Presland, Owen T. Lawrence, David J. Pearton, Leonard M. Milstone, Thomas A. Steitz 
Journal of Investigative Dermatology 
Volume 135, Issue 7, Pages (July 2015)
DOI: /jid
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2. Figure 1
Calcium-independent dimerization and calcium-dependent self-aggregation of profilaggrin N-terminal S-100 fused-type calcium-binding domain. (a) Gel filtration (GF) of PF-CABD with 1mM EDTA (solid line) produced a single peak (180–191ml) but with 5mM CaCl2 (dotted line) produced multiple high MW aggregates (100–186ml). Void volume (Vo). (b) SDS-PAGE of GF eluted protein (1mM EDTA) within 169–197ml fractions demonstrated PF-CABD protein; none was present in 105ml fraction. (c) SDS-PAGE of GF eluted protein (5mM CaCl2) in 101–169ml fractions demonstrated PF-CABD high MW aggregates eluting earlier than expected. L=loaded sample. (d) Light scattering of PF-CABD with 1mM EDTA (solid lines) demonstrated a single homogenous peak with MW (22,590Da) of a PF-CABD dimer (scattering distribution labeled ‘D’) but with 5mM CaCl2 (dotted lines) demonstrated multiple peaks of higher than expected MW (scattering distributions labeled 1–3). (e) Increased ANS fluorescence emission for PF-CABD (2μM) in 2mM CaCl2 (dotted line) compared with PF-CABD in 2mM EDTA (solid line) indicated calcium-dependent structural opening of the protein to expose hydrophobic residues. MW, molecular weight; PF-CABD, N-terminal S100 fused-type calcium-binding domain of human profilaggrin.
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
Crystal structure of the N-terminal S100 fused-type calcium-binding domain of human profilaggrin. (a) PF-CABD is biologically a dimer (protein 1, magenta; protein 2, blue with prime symbol). PF-CABD monomer forms a four-helix bundle. (b) The crystal AU contained two PF-CABD dimers (dimer 1, magenta/blue; dimer 2, orange/gray). The tetramer core is composed of four helix IV helices. (c) PF-CABD dimer rotated forward 120° about x axis compared with a to display the antiparallel helix IV plane connected to a schematic of remaining profilaggrin sequence. (d) Molecular surface of hydrophobic pocket in PF-CABD dimer illustrating two hydrophobic pockets (orange color gradient based on the degree of hydrophobicity); polar residues=blue; orientation 40° backward rotation about x axis compared with panel a. (e) Electrostatic surface potential of PF-CABD dimer, demonstrating acidic calcium-binding loops (red) and uncharged pocket (white). Basic residues=blue. (f) Only five residues are conserved in the hydrophobic pocket (labeled, magenta) across S100 fused-type protein family. Non-conserved residues colored blue. Calcium ions=green. AU, asymmetric unit; B, B domain; F, filaggrin units; H, helix; L, interhelical linker; PF-CABD, N-terminal S100 fused-type calcium-binding domain of human profilaggrin.
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4. Figure 3
The profilaggrin S100 domain interacts with the N-terminus of annexin II. Yeast two-hybrid (Y2H) analysis (a and b) demonstrated that the A (S100) domain of profilaggrin interacts with the N-terminus of annexin II. (a) Bait (green) and prey (pink) plasmid combinations were plated on histidine, leucine, and tryptophan-deficient media containing 10mM 3-amino-1,2,4-triazole. Both full length ANXA2 and a truncated N-terminal protein (ANXA2, 1–44) interacted with human and mouse profilaggrin N-terminus, but an ANXA2 protein lacking the first 14 amino acids (ANXA2, ) showed no Y2H signal. (b) Control (leucine and tryptophan-deficient media) plate showing confluent growth of bait/prey combinations. (c) Association of profilaggrin N-terminus and annexin II in vitro is calcium dependent. Epidermal proteins were immunoprecipitated with either profilaggrin B domain (B1) antibody, a p21/WAF1 antibody, or a pre-immune rabbit control. Immunoprecipitated proteins were separated on SDS/polyacrylamide gels and immunoblotted with annexin II antibody. The lanes show immunoprecipitation with the following: B1 antibody, with no additions; B1/Ca, B1 antibody with the addition of 5mM CaCl2; B1/E, B1 antibody with the addition of 5mM EDTA; and p21/WAF1 antibody or pre-immune serum, with no additions. E represents a control epidermal extract to show annexin II.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Profilaggrin N-terminus and stratifin co-localize in human epidermal granular cells. Double label immunofluorescence was performed on fixed adult human skin using antibodies directed against the profilaggrin N-terminus (green) and stratifin (red). Panel a shows that stratifin is expressed throughout the epidermis, whereas profilaggrin is restricted to the granular layer and anuclear stratum corneum. (b) Shown is a vertical section of adult human skin immunolabeled with stratifin (red) and profilaggrin N-terminus (green) antibodies, with nuclei counterstained with DAPI. Stratifin is localized through the cytoplasm in spinous cells, but in the upper granular layer it is concentrated at the cell periphery where it co-localizes with profilaggrin N-terminus (orange labeling, arrows). Profilaggrin is also present in keratohyalin granules in the characteristic granular pattern but shows little or no association with stratifin when present in the granular (profilaggrin) form. (c) Proposed biological functions of a calcium-dependent and calcium-independent protein interaction network for profilaggrin in human epidermis (based on the current study and the previous study by Yoneda et al. (2012)). DAPI, 4′,6-diamidino-2-phenylindole.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions